The present invention relates to an incident-light fluorescence microscope used in observation of the state of a cell or a tissue in the fields of medicine and biology. The present invention particularly relates to an incident-light fluorescence microscope by which the contour of a cell or a tissue is observed by a differential interference observation method or a pupil modulation microscopic method using a modulation contrast (Hoffman modulation contrast) microscope and at the same time the position of, e.g., a substance is detected by an incident-light fluorescence observation method.
The first conventional technique will be described below. Generally, fluorescence microscopes are extensively used in various fields such as medicine and biology to detect, e.g., a fluorescence-labelled protein or gene in a living tissue or cell. Especially in recently years, it is becoming possible to detect the contour of a cell or a tissue by a differential interference observation method and at the same time to detect a fluorescence-labelled protein or gene by an incident-light fluorescence observation method, thereby checking the position of the protein or gene in the cell or tissue.
Unfortunately, in order to simultaneously perform the differential interference observation and the incident-light fluorescence observation, it is necessary to arrange an analyzer (polarizing plate) in the observation optical system, and the light amount loss increases because light passes through this analyzer. This is particularly a problem since a fluorescent image is formed with very weak light. Therefore, intense excitation light must be irradiated on a sample in order to obtain necessary fluorescence. If this is the case, however, fluorescence photobleaching occurs earlier and the sample may be damaged if it is alive.
To solve these problems, the arrangement of a microscope using polarization and the transmittance characteristic of a dichroic mirror which depends upon wavelength and which is capable of simultaneously performing the differential interference observation and the incident-light fluorescence observation is disclosed in "OPTICAL MICROSCOPY FOR BIOLOGY" (Proceeding of The International Conference on Video Microscopy Held in Chapel Hill, N.C. Jun. 4-7, 1989: A JOHN WILEY & SONS, INC., PUBLICATION: Brian Herman, Ken Jacobson), pages 513 to 522. In this arrangement, a dichroic mirror has a function as an analyzer (polarizing plate) only in a specific wavelength region. Since the transmittance is high at wavelengths longer than the specific wavelength, the fluorescent image in this wavelength region does not darken. Accordingly, the differential interference observation and the incident-light fluorescence observation can be simultaneously performed with a high efficiency.
The second conventional technique will be described below. Recently, it is becoming possible to detect the state of a cell or a tissue by a pupil modulation microscopic method using, e.g., a modulation contrast microscope and at the same time detect a fluorescence-labelled protein or gene by the incident-light fluorescence observation method, thereby checking the position of the protein or gene in the cell or tissue and also checking the movement of the protein or gene.
FIG. 12A shows an arrangement disclosed in Jpn. Pat. Appln. KOKAI Publication No. 51-128548 which is obtained by adding elements for modulation contrast observation to a common microscope. As shown in FIG. 12A, a modulator 102 is arranged on the exit pupil plane of an objective lens 103. This modulator 102 is a filter having regions with three different transmittances as shown in FIG. 12B. In this filter, reference numeral 108 denotes a dark portion (D) which transmits almost no light; 109, a gray portion (G) with a transmittance of approximately 15%; and 110, a bright portion (B) which completely transmits light.
A slit 106 is arranged on the front focal plane of a condenser 105. P1 added to one side of the slit 106 and P2 arranged below P1 are polarizing plates. The quantity of light transmitted through the slit 106 can be controlled by rotating the polarizing plate P2. Also, the exit pupil plane of the lens 103 on which the modulator 102 is arranged and the front focal plane of the condenser 105 on which the slit 106 is arranged are conjugated. Accordingly, an image of the slit 106 is formed on the surface of the modulator 102.
Similar to adjusting a ring slit of a phase contrast microscope to a ring of a phase plate, an operator previously adjusts the slit image to the gray portion (G) 109 on the modulator 102 while monitoring the modulator 102 on the exit pupil plane of the objective lens 103 by using a telescope.
FIG. 13 shows a process in which light transmitted through a transparent phase object forms an image with contrast via a modulator. This principle was announced by R. Hoffman and L. Gross in 1975.
Assume that a transparent sample 115 having a phase distribution forms the shape of a prism as shown in FIG. 13. When light rays passed through the slit 117 and condensed into parallel light rays by a condenser 116 enter this prism, a light ray passing through the inclined portion on the left side of FIG. 13 bends to the left from the incident light rays, and a light ray passing through the inclined portion on the right side of FIG. 13 bends to the right from the incident light rays. A light ray passing through a central portion of the prism where the upper and lower surfaces of the prism are parallel travels straight without bending.
These light rays pass through an objective lens 113 and enter a modulator 112. The light on the left side of FIG. 13 becomes dark light because the intensity of the light is decreased when the light passes through a dark portion (D) 121 of the modulator 112. This dark light reaches an image formation position 124 of the objective lens 113 and forms an intermediate image. The light passing through the central portion passes through a gray portion (G) 122 of the modulator 112 and forms an intermediate image as light having a slightly attenuated intensity. The light on the right side passes through a bright portion (B) 123 of the modulator 112 and forms an intermediate image with no decrease in the brightness. In this manner the image of the transparent prism sample 115 having a phase distribution is formed as a visible image 111 having bright and dark portions in accordance with changes in the inclination and thickness. The foregoing is the principle of the Hoffman modulation contrast observation.
A conventional combined microscope which is the combination of the modulation contrast microscope and the incident-light fluorescence microscope described above will be described below.
FIG. 14 shows the arrangement of the conventional combined microscope. Light emitted from a transmission light source 131 is bent by a mirror 134, passes through a slit 136 analogous to the slit 106 described above, and is guided to a sample 138 by a condenser 137. The light transmitted through the sample 138 is collected by an objective lens 139 and passed through a modulator 140 similar to the modulators 102 and 112 described above. The light is transmitted through a dichroic mirror 141 for incident-light fluorescence and an absorption filter 142, an image of the light is formed by an image forming lens 143, and the image is guided to an eyepiece 145. The foregoing are the illumination and observation optical paths in the modulation contrast method.
At the same time, the following incident-light fluorescence optical path is formed. Light emitted from an incident light source 146 is guided to an excitation filter 151 through a collector lens 147, etc. The excitation light passing through the excitation filter 151 is reflected by the dichroic mirror 141, passes through the modulator 140 and the objective lens 139, and is irradiated on the sample 138.
The fluorescence emitted from the sample 138 is collected by the objective lens 139 in the same manner as for the image formed by the modulation contrast method and passes through the modulator 140. The light is transmitted through the dichroic mirror 141 and the absorption filter 142, an image of the light is formed by the image forming lens 143, and the image is guided to the eyepiece 145.
By the simultaneous use of the two optical paths formed as above, i.e., the optical path formed by the modulation contrast method and the incident-light fluorescence optical path, the two images formed by the modulation contrast method and the incident-light fluorescence observation method overlap each other and can be observed in the eyepiece 145.
In the first conventional technique described previously, the differential interference observation and the incident-light fluorescence observation can be simultaneously performed with a high efficiency. However, the wavelength region for the differential interference observation formed by a bandpass filter is transmitted through a dichroic mirror and an absorption filter. The wavelength region of the bandpass filter is set between the wavelength region of the excitation filter and that of the absorption filter. Therefore, if a fluorescent dye has a fluorescent wavelength close to the exciting wavelength, the excitation light readily mixes in the observation optical system in the above arrangement. This significantly decreases the contrast of the fluorescent image, i.e., the intensity ratio of the fluorescence to the background.
On the other hand, the second conventional technique described above uses the combination of the modulation contrast microscope and the incident-light fluorescence microscope and can detect, e.g., a fluorescence-labelled protein or gene while detecting the state of a cell or a tissue, thereby checking the position or movement of the protein or gene.
Since, however, optical members such as a modulator are inserted in the observation optical system, the brightness of the fluorescent image critically decreases particularly in the incident-light fluorescence observation. In addition, a cell or a tissue observed by the pupil modulation microscopic method using, e.g., the modulation contrast microscope generally has a white background. Accordingly, if the fluorescence to be simultaneously observed has a similar color, it is difficult to separate the image of a cell or a tissue from the fluorescent image.